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Rolling circle amplification of micro-RNA samples

a micro-rna and rna technology, applied in the field of molecular biology, can solve the problems of affecting the relative proportions of nucleotide sequences, and gc-rich sequences, and achieve the effect of under-representation of the 5′ ends of products, and poor amplification of pcr nucleotide sequences in a prior cycl

Inactive Publication Date: 2006-07-27
CHILDRENS HOSPITAL MEDICAL CENT CINCINNATI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008] The present invention provides methods and compositions for the amplification of RNA and comparison of RNA expression levels in multiple samples. The invention allows efficient conversion of RNA into circular cDNA templates suitable for rolling circle amplification. Amplified cDNA can then be labeled using any one of a variety of methods. In an embodiment the amplified cDNA is transcribed into amplified RNA.

Problems solved by technology

Additionally, PCR poorly amplifies some nucleotide sequences, such as GC-rich sequences.
With each PCR cycle the inefficient or poor amplification of a nucleotide sequence in a prior cycle affects the relative proportions of the nucleotide sequences in the sample.
However, each round of the in vitro transcription method requires synthesis of double-stranded cDNA.
The use of random primers tends to result in under-representation of the 5′ ends of the products.

Method used

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Examples

Experimental program
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Effect test

example 1

Synthesis of Double-Strand cDNA

[0107] Total RNA was isolated using the Nanoprep™ kit (Stratagene). The (dT)-T7 primer (SEQ ID NO:1) was HPLC purified then treated with kinase. The (dT)-T7 primer was incubated with 100 pg total RNA. A reverse transcription reaction was performed by the addition of 5× First Strand Buffer, 100 mM DTT, 10 mM dNTP, T4gp32, RNase Inhibitor, and Superscript II reverse transcriptase. The dNTP solution contained equimolar amounts of the four nucleotides. The reaction mixture was incubated. Second strand synthesis was performed by the addition of 5× Second Strand Buffer, 10 mM dNTP, DNA Polymerase I, 1 U E. coli RNAse H, E. coli DNA ligase, and T4 DNA polymerase. The reaction mixture was incubated. The reaction mixture was treated with Exonuclease I. The double-stranded cDNA was purified using the Microcon YM-100 system from Millipore.

example 2

Addition of polyA Tails by Terminal Transferase

[0108] 10× buffer, 25 mM CoCl2, 10 mM dATP, and terminal transferase were added to the cDNA such that the concentration of these components in the terminal transferase reaction was 1× buffer, 2.5 mM CoCl2, and 1.0 mM dATP. The reaction mixtures were incubated. The cDNA was purified using the Microcon YM-100 system from Millipore.

example 3

Circularization of the cDNA by a Splint Oligonucleotide

[0109] T tail primer (SEQ ID NO:2), a splint oligonucleotide, was incubated with kinase. The T tail primer annealed to the polyA tailed, double-stranded cDNA. 10× buffer, 1.0 μl 1 mM dTTP, Klenow fragment, and T4 ligase were added to the reaction components. The reactions were incubated. The cDNA was purified using the Microcon YM-100 system from Millipore.

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Abstract

The compositions of the present invention find use in amplifying RNA obtained from subjects, particularly very small RNA samples. The methods allow conversion of RNA into circularized cDNA suitable for amplification by rolling circle replication. The amplified cDNA is then transcribed into RNA resulting in amplified RNA.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of, and claims priority to and benefit of, PCT / US04 / 022997, filed on Jul. 16, 2004, which application claimed priority to U.S. Provisional Application No. 60 / 487,972, filed on Jul. 17, 2003, the disclosures of each are herein incorporated by reference in their entirety.GOVERNMENT GRANT INFORMATION [0002] This invention was made with Government support under NIH Grant No. 1R01DR61916-01. The United States Government has certain rights in this invention.FIELD OF THE INVENTION [0003] The present invention relates to the field of molecular biology, more particularly to the amplification of RNA and expression analysis. BACKGROUND OF THE INVENTION [0004] Expression analysis is critical to understanding normal and abnormal development, disease progression, and even to determining the presence or absence of a disease state. One of the most common methods of expression analysis involves the use of microarrays. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12P19/34C12N
CPCC12P19/34C12Q1/686C12Q2565/501C12Q2531/125C12Q2525/173
Inventor POTTER, S.LIANG, HUNG-CHI
Owner CHILDRENS HOSPITAL MEDICAL CENT CINCINNATI
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